Pancreatic cancer mortality is growing every year, and radical resection is the most essential therapy strategy. It is critical to evaluate the long-term prognosis of individuals receiving radical surgery. CA19-9 is a biomarker for patient recurrence and survival, however obstructive jaundice has a significant impact on this index. Researchers have attempted to modify the index using various modification methods, but the results have been unsatisfactory. In this study, we adjusted CA19-9 values based on clinical stage and bilirubin and found that it provided better prediction than CA19-9 alone in assessing patients.We analyzed over 5 years follow-up records of patients who underwent radical pancreatic cancer surgery between August 2009 and May 2017 in a single center. We investigated the association of risk factors with overall survival (OS) as well as disease-free survival (DFS) after surgery. Threshold values for high-risk features associated with poor prognosis in resectable pancreatic cancer were determined. The hazard ratios of the indicators were eventually examined under the stratification of patients’ clinical stages.A total of 202 patients were involved in the study. The optimum cut-off values for CA19-9 and CA19-9/TB for predicting overall survival were 219.4 (p = 0.0075) and 18.8 (p = 0.0353), respectively. CA19-9>219.4 increased the risk of patient mortality by 1.70 times , and tumor poor differentiation raised the risk by 1.66 times . Based on clinical stage stratification, we found discrepancies in the predictive efficacy of CA19-9 and CA19-9/TB. CA19-9 was a better predictor in clinical stage 1 , whereas CA19-9/TB indications were better in stages 2  and 3 .CA19-9, CEA, and tumor differentiation are predictors for patients with resectable PDAC. CA19-9 values can be adjusted based on clinical stage and bilirubin levels to better predict overall survival in patients with resectable PDAC. CA19-9>219.4 predicted poor survival in individuals in clinical stage 1, whereas CA19-9/TB>18.8 predicted poor survival for individuals in stages 2 and 3. The morbidity and mortality of pancreatic cancer are rising quickly over the world . Non-surMany studies have been undertaken in order to identify the best serum biomarkers for predicting the prognosis of pancreatic ductal adenocarcinoma (PDAC) patients. Previous research has shown that carbohydrate antigen 19-9 (CA19-9) is a biomarker for recurrence and survival . BilirubIn this study, we reviewed the patient data of our hospital and investigated the correlation between indicators and the overall survival (OS) as well as disease-free survival (DFS) of patients in different clinical stages. It is favorable to the future clinical application of these indicators in order to improve the surgeon’s ability to predict PDAC patients prior to surgery. In compliance with the STROBE reporting checklist, we provide the following article .Between August 2009 and May 2017, we analyzed the data of PDAC patients who underwent radical surgical treatment at our institution . The hospital’s medical record system was used to collect patient information, laboratory and pathological features for this study. All patients did not get preoperative pretreatment but received postoperative adjuvant chemotherapy (based on S1 or gemcitabine). The patient’s latest follow-up was in April 2022. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Ethics Committee of West China Hospital of Sichuan University, and written informed consent was obtained from all patients before surgery.Eligibility criteria: (I) patients with PDAC who underwent radical surgical treatment between August, 2009 to May 2017; (II) no restriction was imposed on age and gender; (III) histopathological diagnosis of pancreatic ductal adenocarcinoma with intact pathological samples.Exclusion criteria: (I) metastasis was found at the initial operation; (II) R2 resection; (III) data on clinical, laboratory characteristics, treatments, outcomes and follow-up are not available; (IV) died within 30 days; (V) lost to follow-up within two years after operation.The OS was defined as from the dates of surgery to the dates of death. The DFS was calculated from the interval between the dates of surgery and the first recurrence or metastasis. If there was no recurrence or metastasis at the time of the patient’s death, the DFS and OS dates were the same.We investigated the correlation between risk factors and the prognosis of patients in different clinical stages. The potential indicators such as age, gender, pain, degree of tumor differentiation, vascular invasion, nerve invasion, cutting edge status, tumor site, CA19-9, Carcinoembryonic antigen (CEA), total bilirubin (TB), CA19-9/TB, and clinical stage, etc. were used to identify the univariate risk factors affecting resectable PDAC patients’ OS and DFS, respectively. The eighth edition of the American Joint Committee on Cancer (AJCC) guidelines for pancreatic cancer is used to determine Tumor-Node-Metastasis (TNM) and clinical stage of malignancies .On the basis of univariate test, Cox survival regression analysis was further carried out to find the risk factor and the hazard ratio (HR). The analysis is based on the premise that the Kaplan–Meier survival curve does not cross. We only investigated variables that were statistically significant after initial screening, on which HR values and P values were built.We performed Pearson correlation to examine the relationship between CA19-9 and tumor pathological parameters such as tumor maximum diameter, lymph node metastases, tumor site, differentiation, vascular invasion, and nerve invasion in order to improve the accuracy of CA19-9 in predicting OS. In addition, a linear regression analysis was applied to see if the indicators and CA19-9 have a linear correlation.Continuous variables were stratified using the X-tile software   and the positive lymph nodes (P = 0.033) according to linear regression analysis. There is no multicollinearity between the two indicators (variance inflation factor (VIF) = 1.000). The Dubin Watson (DW) test was used to determine that the independent variables had no autocorrelation . Based on the linear correlation between CA19-9 and tumor information, the correction formula CA19-9 = 160.562+46.685*d+39.654*ln  was developed.According to the findings, tumor stage has a significant impact on patients’ overall survival. We divided patients into three groups based on their clinical stage to reduce the interference of tumor stage. The ability of CA19-9 and CA19-9/TB to predict disease progression was then tested at different stages. In clinical stage 1, the HR of CA19-9 was higher than CA19-9/TB , while lower in stage 2  and stage 3.In this study, we first looked into the factors that affect OS and DFS, and found that CA19-9, CEA, and low-differentiation were mostly predictors for poor patient outcomes. Meanwhile, using correlation analysis, we found that CA19-9 had a possible linear association with tumor dimensions and the positive lymph nodes. CA19-9 levels may be connected to tumor growth or metastatic burden, according to this research. Despite the fact that this linear association is modest, it gives us fresh ideas. These two indications are, interestingly, closely tied to TNM staging. We hypothesized that the patient’s TNM stage could be effective in predicting the indicators’ predictive effect. We divided patients into groups based on their clinical stage based on this idea. CA19-9 and CA19-9/TB were investigated for their prognostic accuracy in range of clinical stages. CA19-9 was found to have better predictive qualities for patients’ OS when stratified by clinical stage and adjusted with TB. Briefly, we found using CA19-9>219.4 in clinical stage 1 and CA19-9/TB>18.8 in clinical stages 2 and 3 has a better effect on dividing patients’ OS as well as DFS and indicates a worse prognosis. More importantly, TNM staging is broadly assessable from pre-operative radiographic indicators. It is helpful for clinicians to make preoperative judgments on patient prognosis.via pathology. However, predicting a patient’s survival before surgery is challenging. Using CA19-9 to predict the long-term prognosis of resectable patients is a convenient and efficient method   . To redu-1) . Xu et a-1) . It is a-1) , 16. We Other clinical and pathological variables, such as pain, cutting edge status (R0 vs. R1), vascular invasion, and nerve invasion, were found to be of limited utility in assessing prognosis in our study. Pain, as demonstrated by Xu et al., cannot be used alone to predict PDAC . The twoThe limitation of this study is that it was a retrospective study conducted at a single center. Since the cut-off value only comes from the population in this study, the cut-off value mainly shows the trend in the sample data. A bigger sample size is required for an accurate cut-off value. The time span is long, and the level of experience of surgeons varies. Patients were mostly from 5 years ago, therefore current adjuvant and neoadjuvant therapy were not used. In additional to this, we did not consider CA19-9-Low&Lewis (+) pancreatic cancer patients, which may have an uncertain impact on accuracy . FurtherThrough this study, we found that CA19-9, CEA and tumor low-differentiated are markers of a poor prognosis in resectable PDAC patients. CA19-9 has a modest linear correlation with tumor maximum diameter and positive lymph nodes. Following clinical staging, we used CA19-9 to assess clinical stage 1 patients and CA19-9/TB to assess stage 2,3 patients, respectively. CA19-9>219.4 or CA19-9/TB>18.8 suggested a poor long-term prognosis for patients.CA19-9, CEA and tumor low-differentiated are predictors that affect the prognosis of resectable PDAC patients. CA19-9 values adjusted with clinical stage and bilirubin could better predict overall survival in patients with resectable PDAC. For patients in clinical phase 1, CA19-9>219.4 indicates a worse chance of survival. CA19-9/TB>18.8 predicts a worse OS in patients in stages 2 and 3. This study will help clinicians comprehend patients with high preoperative CA19-9 levels and simply probability of patient survival.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by West China Hospital of Sichuan University. The patients/participants provided their written informed consent to participate in this study.ZWW, PZ, CW, and BT conceived and designed the study. ZWW, PZ, XH, and ZHW were responsible for the collection and assembly of data, data analysis, and interpretation. ZWW, ML, and LW were involved in writing the manuscript. ZWW and BT revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any funding including commercial or financial relationships that could be construed as a potential conflict of interest.The authors thank all medical staff who contributed to the maintenance of medical records and access to follow-up records.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A. pretiosum strain metabolism and AP-3 yield, metabolomics analysis was carried out by detecting strain intermediate metabolites at various stages under different dissolved oxygen levels. Moreover, differential metabolite screening and metabolic pathway enrichment analysis were combined to exploit the effect mechanism of soybean oil on AP-3 production. Results suggested that primary metabolic levels of the TCA cycle and amino acid metabolism increased with the increase in dissolved oxygen level, which was beneficial to the life activities of bacteria and the synthesis of secondary metabolic precursors, thus increasing the production of AP-3.A novel approach involving exogenous oxygen vectors was developed for improving the production of biosynthetic Ansamitocin P-3 (AP-3). Four types of oxygen vectors including soybean oil, n-dodecane, n-hexadecane, and Tween-80 were applied to explore the effect of exogenous oxygen vectors on AP-3 yield. It was observed that soybean oil exhibited a better ability for promoting AP-3 generation than the other three oxygen vectors. Based on the results of the single-factor experiment, response surface methodology was employed to obtain the optimal soybean oil addition method. The optimum soybean oil concentration was 0.52%, and the addition time was 50 h. Under this condition, the yield of AP-3 reached 106.04 mg/L, which was 49.48% higher than that of the control group without adding oxygen vectors. To further investigate the influence of dissolved oxygen on precious orange tufts actinomycetes variety  Nocardia sp., and this group of new components presented strong antitumor characteristics [Maytansine has attracted tremendous attention due to its significant antitumor effect , 2. Neveeristics . Differeeristics –8. The aeristics –13. In 2A. pretiosum and the miracle fasciculus actinomycetes A. mirum. Nowadays, increasing the yield of AP-3 is still a hot research area because of the low production of AP-3 fermentation. To promote the biosynthesis of AP-3, researchers have adopted various strategies including mutant screening, medium optimization, and genetic modification [The commonly used strains for biosynthesizing AP-3 include the precious orange tufts actinomycetes variety fication –18. It ification . Since ffication .S. cerevisiae [S. cerevisiae growth and adenosine-methionine synthesis. To increase the available dissolved oxygen, Ciobanu et al. utilized 2% n-dodecane as the oxygen vector [β-galactosidase could be enhanced. To the best of our knowledge, few studies have specifically focused on investigating the effect of oxygen vectors on the production of biosynthetic AP-3.In the process of industrial fermentation of aerobic microorganisms, the level of dissolved oxygen in the fermentation broth is one of the key factors affecting the product yield. The formation of mycelium is able to increase the fermentation broth viscosity, resulting in a rapid decrease in the dissolved oxygen level. Increasing the rotational speed and utilizing pure oxygen can solve the problem of oxygen transfer restriction. Nevertheless, the shear force caused by high stirring and ventilation has adverse effects on mycelium morphology and product yield. In addition, increased ventilation can lead to higher energy consumption. It is necessary to develop an effective way to improve the oxygen transfer rate during aerobic microbial fermentation. The oxygen transfer rate from the gas phase to microbial cells can be improved by adding certain organic liquids to the fermentation broth. These liquids are commonly referred to as oxygen vectors and include n-alkanes, n-fluorocarbons, esters, fatty alcohols, and oils –26. Sincrevisiae . The resn vector . The proPenicillium species. Phenotypes were effectively identified and classified by integrating efficient analytical methods, data processing techniques, and intelligent screening [Metabolomics is an emerging field of systems biology that aims to comprehensively analyze metabolites of cells at certain times and under certain environmental conditions . It has creening . Dallugecreening . Althougcreening .To take advantages of both oxygen vectors and metabolomics, herein, the oxygen utilization efficiency of AP-3 production strains was improved by utilizing an oxygen vector and the effect mechanism of dissolved oxygen on AP-3 biosynthesis was investigated via metabonomics analysis . The infA. pretiosum strain B24-13 was obtained by UV-induced mutation. Before the fermentation process, the basal medium was prepared. The basal medium composition was as follows: saccharose 2.5 g/L, glycerin 1.5 g/L, corn plasm 2.5 g/L, isobutanol 0.2 g/L, calcium carbonate 0.7 g/L, magnesium sulfate heptahydrate 0.05 g/L, L-valine 0.05 g/L, and iron sulfate heptahydrate 0.001 g/L. The spores were germinated, and the cells were grown at 28°C in an incubator shaker, at 220 rpm, for 7 days.The precious orange tufts actinomycetes variety Four types of oxygen vectors including soybean oil, n-hexadecane, n-dodecane, and Tween-80 were investigated in this experiment. Soybean oil and Tween-80 were directly sterilized, and n-hexadecane and n-dodecane were filtered for sterilization. A shaker fermentation medium without oxygen vectors was used as the control. The initial addition concentration was 1% (v/v), and each group performed 3 parallel experiments. After fermentation, the optimal oxygen vector promoting AP-3 biosynthesis was selected by detecting the dry weight of bacteria and AP-3 production in each shaker. After screening the optimal oxygen vector, oxygen-vector concentrations of 0%, 0.5%, 1.0%, 2.0%, and 4.0% (v/v) were examined and 3 parallel experiments were set up in each group. Furthermore, the oxygen vector with the optimal concentration was designed to be added at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h after inoculation and culture. After fermentation, the optimized addition time of the oxygen vector was explored via the detection of dry weight and AP-3 yield of bacteria. Based on the results of the single-factor optimization experiment, RSM was applied to optimize the addition process of the oxygen vector by choosing AP-3 production as the testing parameter. Design-Expert software was utilized to carry out experimental design and data analysis of the optimal oxygen-vector addition program. The response surface model was further verified by investigating the reliability and accuracy of the optimal oxygen-vector additive process optimized by the model.18 column 250 × 4.5 mm, 5 μm) with 70% methanol as the mobile phase. The mobile phase flow rate was 0.8 mL/min, and the analysis temperature was 25°C. The HPLC system was equipped with a PDA detector. AP-3 was detected at 254 nm. Moreover, metabolite analysis was performed by using HPLC-MS  0.1% formic acid solution and (B) 0.1% formic acid-acetonitrile solution, at a flow rate of 0.35 mL/min). For sample pretreatment, about 1 mL of the culture was pumped into precooled methanol-water solution (V : V = 4 : 1) with a rapid sampler within 0.2 s, and then, 200 μL of precooled chloroform was added. The mixture was crushed by ultrasonic method in an ice bath and transferred to the EP tube. Subsequently, 20 μL of internal standard  was added, and the sample was put into vials and dried. After being dissolved in 200 μL methanol-water solution, the sample was stored at −20°C for 2 h and then centrifuged. The supernatant of 150 μL was filtered through an organic filter and transferred to a sample vial, and metabolites were detected by LC-MS. For MS assay, an electrospray ion source was chosen and MS was performed in the positive or negative ionization mode. The experiments were repeated three times, and the average value of measured parameters was utilized in calculations. For dry cell weight detection, the centrifugated bacteria were washed twice with distilled water and then dried in an oven at 60°C and the mass gain was measured.The AP-3 amount accumulated during fermentation was analyzed by the HPLC approach  = 0.0004) also suggested a high significance for the regression model and verified the adequacy of the quadratic model. The regression coefficient of this fitting result was 0.94 (R2), indicating that the fitting data were in good correlation with the monitoring data. As seen in Based on single-factor tests, the RSM was used to optimize the addition strategy of soybean oil. As presented in onducted . The fitAfter 48 h from the start of fermentation, AP-3 production increased obviously and the dry cell weight increased rapidly because of the addition of soybean oil Figures . The resAll metabolites obtained were analyzed qualitatively and quantitatively. Positive and negative models were developed for screening 3765 and 1709 metabolites, respectively. To obtain the pathway information with high genetic similarity with strain B24-13, the identified metabolites were matched with the KEGG database. The related metabolites were screened, including (1) amino acids such as tyrosine (Tyr), proline (Pro), and arginine (Arg); (2) intermediate metabolites in the glycolysis pathway such as glucose-6-phosphate (G6P), 3-phosphoglyceride (3PGA), and pyruvic acid (Pyr); (3) organic acid in the tricarboxylic acid (TCA) cycle such as citric acid, ketoglutaric acid (Akg), and oxaloacetic acid (Oaa); and (4) precursor substances in the Ap-3 biosynthesis process such as UDP-glucose (UDPG) and methoxymalonyl-ACP. These differential metabolites almost cover the AP-3 biosynthesis pathway, facilitating the investigation of intracellular states of strain B24-13 at different dissolved oxygen levels for producing AP-3. To further explore the differences between metabolites, more work still needs to be focused on analyzing the enrichment pathway of differential metabolites.P value ≤ 0.05) of strain B24-13 differential metabolites in different fermentation stages was carried out , succinic acid-CoA, and oxaloacetic acid (Oaa) were significantly upregulated during the stationary phase of fermentation. These metabolites could be converted to precursors for AP-3 biosynthesis. These results highlighted the critical influence of oxygen vectors on the efficiency of biological processes and the metabolic behavior of host cells, which directly affected the production of metabolites.The relative content of pyruvate (Pyr) in the EMP pathway was significantly upregulated. On the one hand, Pyr came into contact with amino acid metabolic pathways or lactic acid. On the other hand, Pyr went into TCA cycle metabolism through acetyl-CoA. Therefore, changes in pyruvate concentration might reveal changes in dissolved oxygen levels in the fermentation environment, which played an important role in metabolic regulation. Moreover, the intermediate products of the TCA cycle including As shown in Bacillus subtilis NX-2 fermentation process [UDP-glucose (UDPD) was the important precursor of AP-3 biosynthesis, and it could be converted to glucose 1-phosphate (G1P) by using glucose 6-phosphate mutase (PGM). Subsequently, G1P was converted to UDPD and produced 3-amino-5-hydroxyl-benzoic acid (AHBA) precursor through the amino-shikimate route. It was observed that the phosphorylation level was significantly increased and the relative content of UDPD was significantly upregulated when the dissolved oxygen level was high. During the AP-3 rapid synthesis period, the UDPD concentration decreased rapidly because of synthesizing AP-3. This was similar to the effect of adding oxygen vectors on metabolic flux distribution in  process . As a prpretiosum strain B24-13. By promoting oxygen delivery, oxygen utilization, cell growth, respiration, metabolism, and AP-3 biosynthesis yield might be improved. The recombinant plasmid pET28a-VGB was transformed into E. coli BL 21, and the expression level of VHb protein was analyzed by using SDS-PAGE. Unfortunately, no transformant was obtained when recombinant plasmid pIJ86-VGB was constructed for protoplast transformation and electrotransformation. The conditions of the transformant need to be further explored. In addition, the recombinant plasmid PIB139-VGB was constructed and the conjugation transformation condition was investigated. The optimal medium was determined to be the MS medium. The concentration ratio of the donor and recipient was 1 : 1, and the best coverage period of the antibiotic solution was 18 h. More effort needs to be put into screening positive zygotes.To further improve the yield of AP-3, the genetic performance of strain B24-13 was improved via genome shuffling technology. In this work, three strategies including PEG-mediated protoplast transformation, electrotransformation, and conjugation transformation were applied to introduce the exogenous VGB gene into the A. Four types of oxygen vectors including soybean oil, Tween-80, n-dodecane, and n-hexadecane were utilized to improve AP-3 yield. Results suggested that soybean oil was the optimum oxygen vector. As the concentration of the oxygen vector was an important factor in the AP-3 biosynthesis process, the impact of different concentrations of soybean oil on AP-3 production was investigated in this research. The optimized addition concentration of soybean oil was 0.5%, and the addition time was 48 h. Furthermore, the optimal addition strategy of soybean oil was explored by using RSM based on the single-factor optimization experiment. We found that the optimum addition amount of soybean oil was 0.52% and the addition time was 50 h. Under this condition, the maximum AP-3 production was 106.04 mg/L. In comparison with the control group without adding soybean oil, an improvement of 49.48% was observed. Metabonomics analysis based on LC-MS was carried out to investigate the intermediate metabolites of the strain at various stages at different dissolved oxygen levels. Positive and negative models were developed for quantitatively screening 3765 and 1709 metabolites, respectively. The metabolites associated with precious orange tufts actinomycetes include amino acids, EMP intermediate metabolites, organic acids in the TCA cycle, and precursors of the AP-3 biosynthesis pathway. The enrichment results of metabolic pathways and the fermentation metabolism curve showed that the TCA cycle and amino acid metabolism increased with the increase in fermentation dissolved oxygen level, which promoted the life activities of bacteria and improved metabolic flux of secondary metabolism. In addition, genetic engineering was investigated to obtain oxygen-carrying genes to improve the oxygen utilization rate of strains. Further work needs to be focused on screening desirable zygotes.


S. aureus specific bacteriophages in clinically recognized biopolymers, i.e., chitosan and alginate, to control the release profile of the antimicrobials. The high-titre S. aureus specific bacteriophages were prepared from commercial bacteriophage cocktails. The polymer mixtures with the propagated bacteriophages were then prepared. The stability of the S. aureus bacteriophages in the biopolymer solutions was assessed. In the case of chitosan, no plaques indicating the presence of the lytic bacteriophages were observed. The titre reduction of the S. aureus bacteriophages in the Na-alginate was below 1 log unit. Furthermore, the bacteriophages retained their lytic activity in the alginate after crosslinking with Ca2+ ions. The release of the lytic S. aureus bacteriophages from the Ca-alginate matrices in the TRIS-HCl buffer solution (pH 7.4 ± 0.2) was determined. After 72 h—0.292 ± 0.021% of bacteriophages from the Ca-alginate matrices were released. Thus, sustained release of the lytic S. aureus bacteriophages can be ensured.The use of implant materials is always associated with the risk of infection. Moreover, the effectiveness of antibiotics is reduced due to antibiotic-resistant pathogens. Thus, selecting the appropriate alternative antimicrobials for local delivery systems is correlated with successful infection management. We evaluated immobilization of the  Pseudomonas aeruginosa osteomyelitis after a femoral fracture [Biomaterials play a crucial role in modern medicine. The goal of using a variety of biomaterials is to restore or maintain the function and shape of a specific part of the human body. However, biomaterials-associated infections (BAIs) are one of the leading causes of implant failure. Moreover, biomaterials commonly serve as a colonization surface for bacteria to form a biofilm. Thus, the formation of a biofilm plays a pivotal role in the etiology of BAIs . As a refracture .6 PFU/mL) during the first 6 h after surgery [Bacteriophages are viruses that infect bacteria and use bacterial cells as hosts for their replication. Several characteristics of bacteriophages, including their ability to self-replicate and self-limit in their hosts, and their high specificity to species of bacteria, make them attractive candidates as therapeutic tools ,12,13. B surgery . However surgery . DeliverAccording to the literature, most studies related to combining bacteriophages with materials, namely encapsulation, immobilization, etc., have been carried out in the food industry ,20,21. AStaphylococcus aureus (S. aureus) is one of the leading causes of skin, soft tissue, bone, and joint infections [S. aureus has become the primary pathogen of increased importance due to its antibiotic resistance. Although drugs such as trimethoprim-sulfamethoxazole and vancomycin are still highly active against most S. aureus strains, resistance to vancomycin has already been reported, raising concerns about the possible reduction or loss of activity of this antibiotic soon [S. aureus infections worldwide, bacteriophages with anti-staphylococcal activity have received increased attention and awareness. Moreover, several reports on these bacteriophages have been published, including their genome analysis, host range traits, and activity [fections ,31. S. atic soon . Due to activity ,34,35,36S. aureus.Bourdin et al. (2014) have reported that pathogen coverage is dependent on the geographical area and epidemiological context and might reduce the effectiveness of commercially available phage cocktails . Thus, fStaphylococcus (S. aureus), Streptococcus , E. coli , P. aeruginosa, and Proteus (P. mirabilis and P. vulgaris) species. The Pyo Bacteriophage cocktail is used for the treatment and prophylaxis of purulent-inflammatory and enteric infectious diseases caused by the above-listed microorganisms. In turn, the Staphylococcal Bacteriophage cocktail leads to specific lysis of bacteria belonging to the S. aureus species. The Staphylococcal Bacteriophage cocktail is used in the treatment and prophylaxis of staphylococcal infections.Two commercially available therapeutic bacteriophage cocktails, namely the Pyo Bacteriophage cocktail  and the Staphylococcal Bacteriophage cocktail , produced by Eliava BioPreparations Ltd.  were used. According to the manufacturer’s information, the Pyo Bacteriophage cocktail leads to specific lysis of bacteria belonging to S. aureus (ATCC 25923)) and incubated overnight at 37 °C. Furthermore, the 3–5 bacterial colonies grown on the plate were inoculated in a liquid medium ) and incubated overnight at 37 ± 1 °C.Culturing of the host bacterial strain. A solid agar plate was inoculated with the host strain  was transferred to each test tube, and the obtained mixture was poured onto a solid TSA plate. After the top agar was solidified, the plates were inverted and placed in the incubator overnight at 37 ± 1 °C. The number of plaque-forming units per mL (PFU/mL) was counted.3) treatment for 2 h at 4 °C, the removal of bacterial debris using centrifugation at 6000× g for 15 min at 4 °C, and filtration using a 0.20 μm filter were performed. The supernatant was then collected, and the final concentration (titre in terms of plaque-forming units per millilitre (PFU/mL)) was determined using plaque assay according to Equation (1):Propagating commercial bacteriophage stocks to obtain higher titre bacteriophage lysates. After plaque assay testing, the webbed plates were selected and flooded with 5–7 mL of TSB. The supernatant and soft-overlay agar was selected. Afterwards, a 2% chloroform (CHClThe procedure of preparing bacteriophage solutions before incorporating them into biopolymers included the following stages:S. aureus bacteriophages was reached.Stages 2 and 3 were repeated until the appropriate titre of the wt/vol) chitosan solution, a commercial chitosan  was dissolved in a 1.0%(vol/vol) acetic acid  aqueous solution at ambient temperature.To prepare the 0.5%(wt/vol) Na-alginate solution, a commercial Na-alginate  was dissolved in deionized H2O under continuous stirring at ambient temperature overnight.To prepare the 1.0%(wt/vol) chitosan and 1.0%(wt/vol) Na-alginate solutions were steam-sterilized in an autoclave Elara 11  at 121 °C for 20 min (no drying). The propagated lytic bacteriophage cocktails were mixed with the sterilized biopolymers solutions at a volume ratio of 1:1.Before adding the bacteriophage solutions, the prepared 0.5% were transferred into the weighing “boats”  and stored at 40 ± 1 °C until the solvent completely evaporated (up to 48 h). The films were then removed from the weighing “boats”.2  solution. The Ca-alginate spheres were obtained by positioning the end of the syringe, with a needle diameter of 1.07 mm, within 90° and around 5 cm above the surface of the 250 mM CaCl2 solution and adding the Na-alginate solution dropwise to the CaCl2 solution under mild stirring. The resulting alginate gel droplets were stirred for an additional 30 min, then strained to remove the “excess” CaCl2 solution.Preparation of the Ca-alginate hydrogel beads  involved crosslinking of the Na-alginate solution (without/with bacteriophages) with the CaCl−1 with a spectral resolution of 4 cm−1 using an attenuated total reflectance module ; 50 scans were averaged. Before every measurement, a background spectrum was taken and deducted from the sample spectrum.The molecular structure of the chitosan and Na-alginate films and the Ca-alginate hydrogel dried beads (with a diameter of 1.5–1.8 mm) (with/without bacteriophages) was analysed using a Fourier-transform infrared spectrometer . FT-IR spectra were recorded in the range of 400–4000 cmS. aureus bacteriophages in the solutions was determined by a plaque assay. A bacterial suspension was prepared from the S. aureus reference strain ATCC 25923 according to the McFarland standard with a turbidity of 0.5. The bacteriophage solutions (50 µL) were placed in a tube with 100 µL of the prepared bacterial suspension, and 5 mL TSB was added. After 24 h of incubation in a thermostat , the contents (after dilution with TSB medium) of each tube underwent the plaque assay (as described in the The titre (PFU/mL) of the S. aureus bacteriophages embedded in the Ca-alginate hydrogels, the hydrogels were dissolved in a 0.1 M citrate buffer solution (pH 6.2) containing 0.09 M sodium citrate dihydrate  and 0.01 M citric acid . To the samples, 500 μL of TSB was added and vortexed until a homogenous solution was obtained. Then, the plaque assay was executed to determine the titre of bacteriophages. Serial dilutions of the bacteria and the sample mixtures were made. Acquired mixtures were transferred onto TSA plates. After an overnight incubation period at 37 ± 1 °C, plates containing 10–100 plaques were considered for further titre calculation according to Equation (1). The encapsulation efficiency of the bacteriophages in the Ca-alginate was calculated according to Equation (2):To determine the number of The results are presented as the mean (of three compositions) encapsulation efficiency ± standard deviations (SD).S. aureus bacteriophages, the Ca-alginate samples were incubated in a tris(hydroxymethyl)aminomethane-hydrochloric acid (TRIS-HCl) buffer solution. The TRIS-HCl solution was prepared according to the EN ISO 10,993–14:2001, by dissolving 13.25 g tris(hydroxymethyl)aminomethane  in 500 mL deionized water under agitation and buffered at pH 7.4 ± 0.2 at 36.5 ± 0.5 °C with 1 M HCl , and filled up to 1 L. Samples were placed in microcentrifuge tubes and poured over with TRIS-HCl pre-heated to 37 °C (1 mL per 0.5 mg sample). The microcentrifuge tubes containing TRIS-HCl and the samples were incubated in the table-top environmental shaker-incubator at 37 ± 1 °C providing mild orbital shaking at 100 rpm. To evaluate the stability of the hydrogels, the incubated Ca-alginate beads, after specific time points , were strained and dried . Mean mass changes as well as standard deviations (SD) were calculated from three independent measurements. To determine bacteriophage release, the release medium (TRIS-HCl) was refreshed after 1, 6, 24, and 72 h. Three independent samples were analyzed at each time point. The titre of bacteriophages in the TRIS-HCl after incubation of the Ca-alginate hydrogels was determined using the plaque assay. Serial dilutions of the bacteria and the sample mixtures were made. Acquired mixtures with added molten 0.7% TSA were transferred onto TSA plates. After an overnight incubation at 37 ± 1 °C, plates containing 10–100 plaques were considered for further titre calculation in PFU/mL. The titre was calculated according to Equation (1). The results are presented as the mean (of three compositions) encapsulation efficiency ± standard deviations (SD).To determine the stability of the Ca-alginate beads and the release of lytic S. aureus bacteriophages increased from 1.2 × 106 PFU/mL to 3.1 × 109 PFU/mL, whereas in the case of the Staph Bacteriophage cocktail—from 5.6 × 105 PFU/mL up to 1.0 × 109 PFU/mL. The obtained high-titre bacteriophage solutions were further incorporated into the biopolymer matrices. Before preparing each new series of samples, the bacteriophage titre of the stock solution was re-determined, and the new value was used for the relevant calculations.The propagation procedure was crucial in increasing the original titre of the commercial bacteriophage cocktails. In the case of the Pyo Bacteriophage cocktail, the titre of the The molecular structure of the original materials  and biopolymers films was analysed using FT-IR. From FT-IR spectra , it was −1 are wide and extended, indicating a non-linear, branched structure. The C-H bond gives a weak signal at 2930 cm−1. All three spectra have two intensive absorbance bands with maxima at 1595 cm−1 and 1400 cm−1 corresponding to C=O and C-O bonds, respectively [In the FT-IR spectra of the Na-alginate , filectively . All spe−1 characteristic to O-H bonds and low-intensity bands at 2930 and 2872 cm−1 characteristic to C-H bonds were detected in the FT-IR spectra. Stretching vibrations of C=O bonds at 1651 cm−1 and bending vibrations of N-H bonds at 1585 cm−1 represent amide groups and indicate partially acetylated chitosan [−1. In the case of the raw material, the absorbance bands of the amide groups are a doublet (−1) and C-H  bonds also changed; their intensity decreased. However, the characteristic FT-IR absorbance bands of the chitosan films, prepared from the biopolymer solution before and after the autoclaving, match. It can be concluded that the molecular structure of the chitosan is slightly changed during the preparation of the solution. However, autoclaving does not further change the molecular structure of the chitosan. Thus, the chosen sterilization method of chitosan, namely autoclaving the biopolymer’s solution at 121 °C for 20 min, is considered suitable.In the case of commercial chitosan B-I, a brchitosan . Since c doublet B-I. The 2+ ions. FT-IR spectra of the Na-alginate and Ca-alginate films prepared from the autoclaved biopolymer solutions are compared in Na-alginate was cross-linked with Ca2+ ions. Accordingly, hydrogen bonding between O-H functional groups decreases. Moreover, the cations’ charge density, radius, and atomic weight change when Ca2+ ions replace Na+ ions. Hence, this shifting of the C-O absorbance bands from 1400 to 1420 cm−1 occurs, C-O stretching vibration absorption increases, and an absorbance band at 1053 cm−1 appears [−1 that is assigned to the stretching vibration of C-H in the six-membered ring, which cannot be observed in the spectrum of Ca-alginate. Crosslinking with Ca2+ ions promoted the formation of the egg-box model. Thus, the stretching vibration of C-H in the six-membered ring of the Ca-alginate molecule is limited [Absorbance bands attributed to stretching vibrations of O-H bonds for Ca-alginate are lower than for Na-alginate. This is due to the role of O-H and -COO- groups in the formation of a chelating structure with Ca appears . Na-algi−1 correlate with the presence of proteins, polysaccharides, and nucleic acid macromolecules. The range 1200–900 cm−1 is typical for carbohydrates, 1500–1200 cm−1 corresponds to the carboxyl groups, and 1700–1500 cm−1 is characteristic of proteins, and within the 3000–2800 cm−1, the lipid fraction is observed [The molecular structure of biopolymers films after the addition of bacteriophage suspensions was characterized and is shown in observed . The maiThe potential of using the chosen biopolymers  for immobilizing commercial bacteriophage stocks was investigated. First, the interaction of bacteriophages and biopolymer solutions was analysed.S. aureus bacteria due to lysis by bacteriophages (wt/vol) chitosan resulted in a complete loss of titre. This is due to the solution’s acidic nature (pH 4.07). Given this observation, chitosan was not used in further studies, and the emphasis was placed on the effect of Na-alginate on the viability of bacteriophages within commercial stocks. In the case of the Na-alginate solution, bacteriophages retained their lytic activity, which was confirmed by the formed plaques. In the case of the Staph bacteriophage, the plaques were smaller. The size of the plaque is proportional to the efficiency of adsorption, the length of the latent period, and the burst size of the bacteriophage. Also, the bacteriophages with larger heads (a significant fraction of myophages) tend to form smaller plaques compared to the bacteriophages with smaller heads (a significant fraction of sipho- and podophages). This is because larger virions (with larger heads) would diffuse more slowly through the top agar layer than smaller ones, thus resulting in smaller plaques [Qualitatively, the lytic activity was assessed by plaque formation in a lawn of iophages . In the  plaques .Quantitatively, the number of bacteriophages in the Na-alginate solution was compared with the expected number, and a reduction of titre was calculated in each case .Both commercial bacteriophage stocks showed good stability in the Na-alginate solution with the titre reduction of less than 1 log unit. For further studies, the bacteriophage showing higher stability, i.e., with smaller titre reduction, was chosen to sustain high concentration. Thus, further studies were performed using the Pyo bacteriophages. In addition, as shown in The viability of the bacteriophages during drying (at 40 °C) of the Na-alginate was determined, and the results are summarized in 2, i.e., the incorporation of the bacteriophages into the Ca-alginate hydrogel, was below 1 log unit , reaching an embedded phage concentration of 3 × 107 PFU/mL [S. aureus phage K in Ca-alginate microspheres for oral delivery, achieving a bacteriophage encapsulation efficiency of 95% of added 108 PFU/mL [S. aureus bacteriophages into Ca-alginate hydrogels.To compare, Cobb et al. (2019) reported on Ca-alginate injectable hydrogels for the delivery of genetically modified phage vB_EfaS_LM99  observations described in . The bac4 PFU/mL. However, it is difficult to speculate whether such an amount would be therapeutically effective due to the lack of clinical studies on the local supply of bacteriophages using biomaterials. These results suggest that such an approach can ensure a sustained local supply of bacteriophages.A gradual increase in the titre of released bacteriophages was observed for 72 h . After 1S. aureus lytic bacteriophage suspensions were prepared from commercial bacteriophage cocktails  using a propagation procedure. The acidic medium of the chitosan solution is lethal for the bacteriophages. Drying of the Na-alginate is critical for the loss of lytic activity of the bacteriophages. In turn, the bacteriophages retained their lytic activity in the Na-alginate solution, and the titre reduction of bacteriophages was below 1 log unit. Furthermore, the immobilized bacteriophages retained their lytic activity in the Ca-alginate hydrogel, which can serve as bacteriophage delivery systems for the sustained local delivery of antimicrobials. Further biological studies are needed to predict the necessary concentration of bacteriophages in this type of system to achieve a therapeutic effect.The high-titre


In this study, reduced graphene oxide (RGO) with a hollow nanostructure was successfully synthesized by layer-by-layer self-assembly using electrostatic interactions and van der Waals forces between building blocks, and its lithium storage characteristics were investigated. After 800 cycles at a current density of 1 A/g, the microwave-irradiated RGO hollow spheres (MRGO-HS) maintained a capacity of 626 mA h/g. In addition, when the charge/discharge capacity was measured stepwise in the current density range of 0.1–2 A/g, the discharge capacity of the RGO rapidly decreased to 156 mA h/g even at the current density of 2 A/g, whereas MRGO-HS provided a capacity of 252 mA h/g. Even after the current density was restored at a current density of 0.1 A/g, the MRGO-HS capacity was maintained to be 827 mA h/g at the 100th cycle, which is close to the original reversible capacity. Thus, MRGO-HS provides a higher capacity and better rate capability than those of traditionally synthesized RGO. With the development of rechargeable energy storage technology, lithium-ion storage has been widely used in various electric devices ,2. ThereThe graphitization degree, structural disturbance, and porosity characteristics of carbon materials greatly influence the ability and mechanism of lithium-ion intercalation. On this basis, new graphite carbon anodes with disordered structures, which are advantageous for lithium-ion intercalation/deintercalation and for increasing interlayer distances, have been continuously proposed. As it has already been reported, higher lithium storage capacities can be obtained by controlling the layer structure of graphene nanosheet (GNS) materials, and the structural control of nanosheet materials can greatly affect the performance of LIBs . In addiIn this work, by using layer-by-layer self-assembly and subsequent microwave irradiation, reduced graphene oxide RGO with a hollow nanostructure was synthesized with a wider interlayer distance than that of chemically reduced graphene oxide, as seen in GO was synthesized with reference to the modified Hummer’s method . We addeRGO as a control group was prepared using the conventional chemical reduction method using hydrazine . GO (1.0The synthesis process for MRGO-HS is shown in θ). Microstructures, such as the morphology and the interlayer distance of MRGO-HS, were determined using a field-emission scanning electron microscope  and a high-resolution transmission electron microscope (HRTEM)  with a 200-keV acceleration voltage. We also measured the Fourier transform infrared (FTIR)  and Raman  spectra using KBr pellets to analyze the carbon bonds before and after the reduction of the synthesized samples. In addition, we used X-ray photoelectron spectroscopy (XPS)  to analyze the atomic composition of the surface. N2 adsorption-desorption experiments were performed using Autosorb-iQ and Quadrasorb SI instruments , and pre-experiment samples were dehydrated at 100 °C for 12 h using a vacuum oven. Thermogravimetric analysis (TGA) experiments in an ambient condition were conducted using an STA-S1000 analyzer .The crystal structure of the samples was investigated by X-ray diffraction (XRD) using Bruker D2 phaser  with Cu-Kα radiation (λ = 1.54056 Å) in the range of 4° to 70°  test using WMPG1000 , and sweep speeds of 0.1, 0.3, 0.5, and 0.7 mV/s were performed in the voltage window of 0.01–3.0 V. The electrical conductivity and lithium-ion diffusion behavior of the anode materials were analyzed using electrochemical impedance spectroscopy  data. The electrochemical experiments, such as electrochemical performance, CV, and EIS, were analyzed at room temperature.Charge and discharge experiments to investigate lithium-ion storage characteristics were performed using battery test equipment . MRGO-HS, which was used as an active material, was made in the form of a slurry for casting to copper foil. N-methyl-2-pyrrolidone was used for mixing the active material, conductive carbon, and polyvinylidene fluoride in a ratio of 7:2:1. The resulting slurry was cast flat throughout the copper foil. Then, it was dried for 12 h in a vacuum oven at 100 °C. The coin-type half-cell (type 2032) was manufactured in a glove box filled with Ar gas. The battery cell consisted of an active electrode, a 3501-type separator, and a lithium metal counter electrode. The internal electrolyte used 1M LiPFc-direction is not uniform [θ = 23°. The average basal spacing of MRGO-HS was turned out to be 0.39 nm, which is much larger than that of RGO (0.37 nm). It is expected that the enlarged interlayer spacing of MRGO-HS could be quite advantageous for the fast transport of lithium ions, resulting in enhanced electrochemical lithium storage properties.In  uniform . Moreove uniform ,21,22,23The morphological features of MRGO-HS are also shown in the HRTEM images. −1 are shown in the case of the GO spectrum, which imply the existence of C–O, C–O–C, C–OH, C = C, and C = O (carboxylic acid and carbonyl moieties). After the reduction process, the FTIR peak that appeared in GO almost disappeared or decreased greatly [−1 represents C = C and is displayed because of the unoxidized graphene domain. The absorption peak at 1174 cm−1 may correspond to the remaining O–H and C–O stretching. These FTIR data show that the oxygen functional groups present in the GO were removed after the reduction process, which is in good agreement with the XPS data to be discussed in XPS analysis [−1 appearing in the FTIR spectrum of the (d) PS beads and (e) PS/GO core–shell are not observed. This indicates that the PS beads used as template materials can be effectively removed by microwave treatment with an organic solvent. Moreover, when compared with the GO spectrum, all functional groups of MRGO-HS almost disappeared. Therefore, not only the PS beads used as templates were effectively removed using microwave irradiation in an organic solvent but also the oxygen functional groups in GO. greatly ,25,26. Ianalysis ,28,29. I−1 [2g vibrational mode of the sp2 carbon atom with a hexagonal structure. In the GO spectrum, a broad G-band can be seen at 1595 cm−1, and a D-band representing a failure due to structural defects can be seen at 1350 cm−1. This leads to smaller sp2 domains during oxidation. The D-band is related to the disordered A1g breathing mode of the aromatic ring [−1, respectively. MRGO-HS (1.05) also shows a higher ID/IG ratio than that of GO (0.88). Comparing the average sizes of the sp2 domains using Knight’s empirical equation [2 domains in MRGO-HS have smaller than those in GO. Although the size after the microwave reduction process was small, more new graphitic domains were created than those present in GO [ID/IG ratio, which was higher (1.26) than that of MRGO-HS. Therefore, larger sp2 domains could be restored from GO when using microwave reduction rather than chemical reduction [Structural changes in the process of reducing GO can be seen in the Raman spectrum . As alre−1 . The G-btic ring ,32,33,34equation ,37, the nt in GO . Therefoeduction ,40,41,422 framework. In addition, a peak due to epoxy and alkoxy groups is observed at 286 eV, and carbonyl and carboxyl functional groups can be confirmed through the peak at 289 eV. Therefore, it can be known that the oxygen-containing functional groups in GO were bonded to the surface of the graphene layer, confirming the successful synthesis of GO [2 C–C bond of the graphite structure was almost restored by showing that the oxygen functional group was removed at a considerable rate [The XPS analysis was used to examine the chemical composition before and after the reduction of the synthesized samples. The XPSPEAK41 software was used for the deconvolution of the C 1s and O 1s peaks. S-peak-type and Shirley-type backgrounds were used. In addition, parameters such as the full width at half-maximum, peak position, and area that can be adjusted during deconvolution were used, and joints using Lorentzian–Gaussian functions were performed. Comparing the atomic percentage of each sample, the ratio of carbon is further increased in RGO and MRGO-HS compared with that in GO, indicating a higher carbon-to-oxygen (C/O) ratio. is of GO ,44,45. Able rate ,47. In tble rate ,49,50.The oxygen-containing functional groups of each sample were further investigated using the O 1s spectrum in Therefore, the above XPS analysis results show that microwave-assisted reduction can be effectively used to restore the graphite framework from GO.2 adsorption–desorption isotherm measurements. MRGO-HS showed isotherms characteristic of Brunauer-Deming-Deming-Teller-IV-shaped mesoporous materials with H3-type hysteresis loops. This is usually associated with capillary condensation [2 adsorption in low relative pressure (P/P0 < 0.6) is rarely seen, and the adsorption and desorption curves are consistent as adsorption and desorption consist of single-layer adsorption and a fairly reversible process. Conversely, in the relatively high-pressure region (P/P0 > 0.6), it shows a fairly steep adsorption point, and an H3-type hysteresis loop appears. These types of isotherm and hysteresis are mainly manifested by parallel walls or open slits and narrow neck capillaries. Therefore, it can be seen that most of the pores of MRGO-HS are slit-shaped pores slit by the aggregation of nanosheets. In contrast, MRGO-HS showed only weak nitrogen adsorption up to 1-bar relative pressure and no pronounced hysteresis. This is due to the multiple layers of GO filled with oxygen functional groups. The isotherm of GO was classified as BDDT type III, which is characteristic of nonporous materials. The Brunauer–Emmett–Teller specific surface area was 20 m2/g in the case of GO and 80 m2/g for MRGO-HS, confirming the increase in the surface area caused by the microwave-assisted reduction process. In addition, for MRGO-HS compared with GO, the N2 isotherm shows an increased slope of P/P0 between 0.05 and 0.5, indicating the presence of developed mesopores. The pore size distribution of MRGO-HS was calculated using the Barrett–Joyner–Halenda (BJH) method. As shown in the inset of The porosity and surface area of MRGO-HS were studied using Nensation . In MRGORct (charge transfer resistance) was much smaller for the MRGO-HS electrode. Further details are discussed in a previous study [Rct values of the two electrodes decreased significantly. Because of the surface activation, Rct was greatly reduced, and no slick growth of the SEI layer was observed on the two electrodes up to 70 cycles [ct value compared to MRGO-HS. However, after 70 cycles, Rct value of RGO is similar to that of MRGO-HS. This is because both materials consist of similar graphene frameworks. Therefore, when the electron transport channel of the graphene framework is activated by the reversible insertion/deinsertion reaction of lithium ions in RGO, the charge transfer behavior can be increased, similar to that of MRGO-HS. Zre) in the low-frequency region and ω−0.5. This relationship is described by Equation (1).EIS data were measured to confirm the improved conductivity of MRGO-HS. us study . After 70 cycles . BecauseThe line displayed in the low-frequency region is due to the diffusion of lithium ions, also called Warburg diffusion. The smaller the slope of the Warburg plot determined via least-squares fitting, the faster the diffusion of the lithium cations. As shown in + and electrons involved in the electrochemical reaction. In i) to be measured and the scan speed (ν) is expressed by Equations (2) and (3).CV measurements were made at various sweep speeds of 0.1, 0.3, 0.5, and 0.8 mV/s, thereby comparing the dynamics of MRGO-HS and RGO to further investigate their capacitive behaviors. The CV curve at each sweep rate showed a similar shape a,b. The a and b are empirical parameters. As the b value is displayed closer to 0.5, the capacity contribution of the diffusion control process is more dominant. In addition, the closer the value of b is to 1, the greater the surface-controlled current, indicating an ideal capacitive behavior [ν) (mV/s) versus log(i) (mA/g) plot measured at other sweep speeds from 0.1 to 0.8 mV/s. The value of b of the peak at the anode can be obtained from the slope. The b value appears at 0.785, a value greater than 0.5, which means that the storage and release of Li+ are governed by pseudocapacitive and diffusion-controlled Faraday processes [b value of RGO was calculated as 0.755. Therefore, the advantage of the hollow sphere structures in pseudocapacitive process can be seen by comparing the slopes in In the given equations, behavior . Figure rocesses . The b vk1ν in Equation (4), the ratio of the current due to the pseudocapacitive effect is shown, and k2ν0.5 shows the current response by a diffusion-controlled process. k1 and k2 are constant values. Therefore, k can be obtained through the relationship between i (current in the CV curve) and ν (scanning speed), as shown in the following equation [In the case of equation ,64,65,66k at scan speeds of 0.1, 0.3, 0.5, and 0.8 mV/s. We compared the ratio of the MRGO-HS and RGO current due to the pseudocapacitive effect. The contribution ratio of pseudocapacitance increased with increasing scan speed, and the contribution ratios of the pseudocapacitance in MRGO-HS were higher than those in RGO at all measured scan speeds [n speeds . These en speeds . TherefoWe proposed a process for producing MRGO-HS using layer-by-layer self-assembly and subsequent microwave irradiation in organic solvents. As expected, the structure of the hollow spheres showed stable cycling and speed performance compared with those of the conventional RGO and showed improved charge transfer efficiency. In addition, MRGO-HS showed improved pseudocapacitance behavior and clearly demonstrated its structural advantages. Moreover, the superior speed performance associated with the pseudocapacitive effect can be expected in the anodes of next-generation LIBs. Therefore, MRGO-HS with an extended interlayer gap compared with that of existing graphene-based materials can be evaluated as a very good structure for the anode materials of LIBs.


The effects of the focusing geometry, grating order, laser energy and grating length on the spectral characteristics of the PbP FBG were investigated. After optimizing these parameters, a high-quality first-order PbP FBG with a reflectivity > 99.9%  and insertion loss (IL) of 0.03 dB was successfully created. Moreover, taking advantage of the excellent flexibility of the fs laser PbP technology, high-quality FBGs with various Bragg wavelengths ranging from 856 to 1902.6 nm were inscribed. Furthermore, wavelength-division-multiplexed (WDM) FBG arrays consisting of 10 FBGs were rapidly constructed. Additionally, a Fabry-Perot cavity was realized by using two high-quality FBGs, and its birefringence could be reduced from 3.04 × 10−5 to 1.77 × 10−6 by using a slit beam shaping-assisted femtosecond laser PbP technology. Therefore, such high-quality FBGs are promising to improve the performance of optical fiber sensors, lasers and communication devices.We experimentally studied the inscription of fiber Bragg gratings by using femtosecond ( Fiber Bragg gratings (FBGs) have become an indispensable component element in modern optics and information technology since the first FBG proposed by Hill et al. in 1978 ,3,4,5,6.fs) laser direct writing technologies. UV laser-based phase-mask technology is the most conventional method for the fabrication of FBGs, which can offer FBGs with high reflectivity and negligible loss [fs laser phase-mask technology was proposed by Mihailov et al. [fs laser Talbot interferometer methods exhibit good wavelength flexibility for FBG inscription [fs laser, complicated adjustment of the space optical path is required, which shows low efficiency in FBG inscription.To date, three typical approaches are used to inscribe FBGs: the phase-mask technologies, laser Talbot interferometer methods, and femtosecond  can be flexibly controlled [κ/α = 46 (κ is the coupling strength coefficient and α is the scattering loss coefficient). To increase the κ/α, Williams et al. proposed a continuous core-scanning technique [κ/α > 780. Nevertheless, both the LbL and continuous core-scanning technologies are time-consuming in grating inscription. As the most common direct writing technology, point-by-point (PbP) technology was first reported by Marshall et al. [κ/α  and high birefringence due to the highly localized and elliptical RIMs in the fiber core [fs laser beam shaping by a cylindrical lens or a slit was demonstrated to effectively increase the κ/α of point-by-point FBGs (PbP FBGs) [κ/α of 172.46 [The ntrolled . Thus, Fntrolled . The linntrolled , in whicechnique , achievil et al. . This teber core ,17. RecebP FBGs) ,19. We hf 172.46 . Additiofs laser PbP technology to inscribe high-quality FBGs. The effects of focusing geometry, grating order, laser energy and grating length on the spectral response were investigated. After optimizing these parameters, a high-quality first-order PbP FBG with a Bragg resonance attenuation of 37.7 dB and IL of 0.03 dB was successfully created. Additionally, high-quality FBGs with various Bragg wavelengths and WDM FBG arrays were flexibly created by the fs laser PbP technology. Moreover, the birefringence of the FBG Fabry-Perot (FBG-FP) cavity was investigated, and a slit beam shaping-assisted fs laser PbP technology was used, achieving an FBG-FP cavity with a low birefringence of 1.77 × 10−6. The inscribed high-quality FBGs are promising candidates for applications in optical fiber communications, sensing and lasers.In this article, we further developed the fs, and a repetition of 200 kHz is used as the laser source. A waveplate and Glan polarizer are used to adjust the pulse energy and polarization of the laser beam. In particular, a tunable slit is inserted before the objective, which can modify the laser beam to the objective by changing the width of the slit. The laser beam is tightly focused in the core of a coating-stripped single mode fiber  by a high numerical aperture (NA) oil-immersion microscope objective. Index-matching oil is applied between the objective and the interface of the fiber for the purpose of reducing the aberration at the focal region. The fiber held on two clamps is mounted on a computer-controlled three-dimensional air bearing translation stage , ensuring the accurate translation of the fiber during the fabrication process. The microscopic image of the fiber is monitored in real time by a high-resolution CCD. During the FBG inscription process, the shutter is opened and the fiber was translated along the fiber axis , an optical spectrum analyzer  with a resolution of 20 pm. For a uniform FBG, the reflectivity R can be expressed as:κ is the grating coupling strength coefficient, which is determined by the refractive index modulation (Δn), mode overlap factor η, and Bragg wavelength Bλ:The experimental layout of the femtosecond laser micromachining setup for fabrication of the high-quality FBGs is shown in κ and insertion loss (IL) of an PbP FBG are largely dependent on the size, shape and morphology of the refractive index modulations (RIMs) in the fiber core. The Δn is the amplitude (or AC component) of the RIM, which could be controlled by changing the pulse energy and ratio of the modification to space in a grating pitch . Thus, to achieve a high-quality PbP FBG, one could tailor the focusing geometry, grating order, pulse energy and grating length. As for the birefringence of the PbP FBGs, it is highly dependent on the size, ellipticity and position of the RIMs in the fiber core [fs laser beam.The coupling strength coefficient ber core , which cfs laser point-by-point technology depends largely on the focusing conditions. In particular, the NA of the objective plays an important role in the fabrication of FBGs. If an fs laser beam with a central wavelength of λ is focused by a certain NA microscope objective into a fiber, the focused beam parameters can be expressed as:w0 and z0 are the beam waist and Rayleigh length of the focused beam, respectively, n is the effective refractive index of the fiber cladding. Note that both 0w and 0z are increasing as the NA of the objective decreases. That means the size of the RIM induced by fs laser pulse will change with objectives of various NA.The spectral quality of the FBG by fs laser pulses in the fiber core [η is increased with the larger RIM, thus improving the coupling strength coefficient κ of grating.Initially, we experimentally studied the effect of objective NA on the spectral response and RIM morphologies of the PbP FBGs. Herein, two 4 mm-long gratings were inscribed in two sections of an SMF by using two oil-immersion objectives with NAs of 1.40 and 1.25, respectively. The translation speed was set as 1.07 mm/sec and thus two FBGs with the same period of 1.070 μm were obtained. The on-target pulse energies for fabricating the gratings were 70 and 55 nJ, respectively. Note that it only takes ~3.7 s for the inscription of each FBG. To obtain the FBG cross-sectional morphologies, we firstly transversally cleaved the FBG at the grating region by a fiber cleaver . Then, a microscope  with an oil-immersion objective  was employed to observe the cross-sectional images. As a result, the cross-sectional images of the fiber core could be visualized and captured. ber core . In addifs laser with a 1.25-NA focusing, the fs laser focused by the 1.40-NA objective led to a more localized RIM. Thus, for the fabrication of two FBGs with the same grating strength, a higher pulse energy was required to increase the RIM area while using the 1.40-NA objective. Then, an enhanced scattering loss was induced by the higher fs laser pulse energy focused by the higher NA objective [bjective . As a ren. To investigate the effect of grating order, a 4 mm-long grating was inscribed using a translation speed of 0.535 mm/s. Notably, an optimized pulse energy of 38 nJ and a 1.25-NA oil-immersion objective was used to obtain a desired grating strength of 21.67 dB. The top-view morphology and corresponding transmission and reflection spectra are shown in a = 1.22λD/NA, where aD is the diameter of the focal spot, λ is the laser wavelength, and NA is the numerical aperture of the objective. As for the 513 nm fs laser and 1.25-NA objective employed in our experiments, one can calculate that the spot size is ~501 nm. Additionally, due to the effect of nonlinear multiphoton absorption, the spatial resolution of fs laser nanofabrication could be further improved [n. In this case, the RIMs in the fiber core induced by the lower fs pulse energy are smoother, and thus reduce the insertion loss [Next, we investigated the grating order on the FBG spectral response, since the grating order has a significant impact on the RIM Δimproved . Thus, aR of an FBG is expressed in terms of the coupling strength coefficient κ and grating length L, as expressed in equation (2). The IL, resulting from the photo-induced damaged RIMs, is highly correlated to the intensity of the fs pulse energy. Thus, with appropriate control on pulse energy and grating length, one could expect further improvement in spectral quality. Herein, a 40 mm-long grating was inscribed using a translation speed of 0.535 mm/s, leading to a first-order FBG with a period of 0.535 μm and a Bragg wavelength of 1549.2 nm. The pulse energy for inscribing the FBG was set as 19 nJ to obtain smooth RIMs. The spectrum of the inscribed FBG was shown in κ/α is a crucial parameter of high-quality FBGs. The coupling strength coefficient κ and scattering loss coefficient α can be expressed as [BT is the Bragg resonance attenuation, ILT is the insertion loss, and L is the grating length. Significantly, the κ/α of the inscribed 40 mm-long first-order FBG is 1459, which is much higher than the FBGs fabricated by the core scanning method [According to the results and analysis above, the spectral characteristics of FBGs are dependent on the geometry of the RIM region and grating order. Moreover, the reflectivity essed as :κ = ln  and two sections of standard SMF using a translation speed of 0.592, 1.072, and 1.316 mm/sec, respectively, leading to three second-order FBGs  with periods of 0.592, 1.072 and 1.316 μm. Identical pulse energy of 70 nJ was used for inscribing S1-S3. As shown in The Additionally, two WDM FBG arrays with various Bragg wavelengths were successfully fabricated in a thin-core SMF  and standard SMF, respectively. Ten 4 mm-long FBGs  were gradually inscribed into a thin core SMF with translation speeds ranging from 0.575 to 0.584 mm/s with a step of 1 µm/s, achieving a WDM FBG array with 10 Bragg wavelengths ranging from 838 to 856 nm . The spacing between two adjacent FBGs is 2 mm along the fiber axis. A 20 nJ pulse energy was used for inscribing the WDM1. In the same way, another WDM FBG array  with Bragg wavelengths ranging from 1538 to 1562 nm was inscribed in a standard SMF using a 24 nJ pulse energy. fs laser PbP technology exhibit high birefringence, resulting from the laser-induced asymmetric and elliptical RIMs in the fiber core. This property could be exploited for polarization-selection in fiber lasers. For illustration, a FBG Fabry-Perot (FBG-FP) cavity was fabricated in a section of SMF by inscribing two 2 mm-long uniform first-order FBGs with a spacing of 1 mm along the fiber axis. The two inscribed FBGs have the same period of 0.535 μm, and the on-target single pulse energy used for fabricating each FBG was 37 nJ. The polarization-resolved transmission spectra were measured by a Muller-matrix-based commercial polarization analysis system that consists of a tunable laser , a polarization synthesizer , and an optical power meter  with a resolution of 0.3 pm.Generally, FBGs inscribed by using Bn = Δλ/2Λ, where Δλ is the Bragg wavelength difference between two orthogonal polarization modes , Λ is the period of grating. The FBG-FP cavity exhibits a Δλ of 32.58 pm, corresponding to a high ΔBn of 3.04 × 10−5. Moreover, the difference in transmission resonance attenuation between TE and TM modes, denoted by ΔT in fs laser PbP technology. The slit width was set as 0.8 mm and the on-target single pulse energy was 86 nJ. The FBG-FP consisted of two 2 mm-long uniform first-order FBGs with a spacing of 1 mm. λ of 1.9 pm, corresponding to a low ΔBn of 1.77 × 10−6. The difference in transmission resonance attenuation ΔT is ~0.3 dB, and the PDL is as low as 0.81 dB. Compared with the FBG-FP without slit beam shaping, the FBG-FP with slit beam shaping shows a significantly lower birefringence due to the near-circular cross-sectional pattern induced by the shaping fs laser beam. Such a low-birefringence FBG-FP is valuable in the fields of fiber-optic telecommunications and high-accuracy fiber-optic sensing. In addition, the ellipticity of the cross-sectional pattern in RIMs could be further adjusted by changing the slit width. Thus, it is promising to achieve birefringence-tunable FBG-FPs with the slit beam shaping method for fs laser PbP technology.On the contrary, the polarization splitting would be a disadvantage when the FBG-FP cavity serves as a narrow-band filter or a sensor based on the narrow transmission band, since the high birefringence could lead to a higher bit error rate and reduced sensing accuracy. To address this, the slit beam shaping method was used for inscribing an FBG-FP based on the fs laser PbP technology. The effects of the focusing geometry, grating order, laser energy and grating length on the spectral response were experimentally investigated. With the optimized parameters, a 40 mm-long high-quality first-order FBG with a reflectivity >99.9% and insertion loss of 0.03 dB was successfully created. The corresponding ratio of coupling strength coefficient to the scattering loss coefficient is significantly improved to 1459. Subsequently, three FBGs with Bragg wavelengths of 856, 978.6, and 1902.6 nm were obtained, and two low-loss WDM FBG arrays consisting of 10 FBGs were realized, with Bragg wavelengths ranging from 838 3 to 856 nm and 1538 to 1562 nm. Moreover, birefringence-tunable FBG-FP cavities were realized by using a slit beam shaping method for fs laser PbP technology. The birefringence of the inscribed FBG-FP cavity with slit beam shaping was reduced to 1.77 × 10−6. Therefore, the high-quality FBGs created by fs laser PbP technology are promising in the applications of communications, sensing and lasers.We have further demonstrated the inscription of high-quality FBGs with high reflectivity and low insertion loss based on


Tilia spp.) and strawberry tree (Arbutus unedo) nectar compounds. Unedone from A. unedo nectar inhibited the common bumblebee gut parasite Crithidia bombi in vitro and in Bombus terrestris gynes. A compound in Tilia nectar, 1--6-O-β-d-glucopyranosyl-β-d-glucopyranose (tiliaside), showed no inhibition in vitro at naturally occurring concentrations but reduced C. bombi infections of B. terrestris workers. Independent of microbiome status, tiliaside was deglycosylated during gut passage, thereby increasing its antiparasitic activity in the hindgut, the site of C. bombi infections. Conversely, unedone was first glycosylated in the midgut without influence of the microbiome to unedone-8-O-β-d-glucoside, rendering it inactive against C. bombi, but subsequently deglycosylated by the microbiome in the hindgut, restoring its activity. We therefore show that conversion of nectar metabolites by either the host or the microbiome modulates antiparasitic activity of nectar metabolites.Antimicrobial nectar secondary metabolites can support pollinator health by preventing or reducing parasite infections. To better understand the outcome of nectar metabolite–parasite interactions in pollinators, we determined whether the antiparasitic activity was altered through chemical modification by the host or resident microbiome during gut passage. We investigated this interaction with linden (This article is part of the theme issue ‘Natural processes influencing pollinator health: from chemistry to landscapes’. The accurate mass of the m/z 241 pseudomolecular ion [M + H]+ in positive mode analysed via HR-ESI-MS furthermore matched the predicted mass from the molecular formula of the [M + H]+ ion  and had a matching MS2 spectrum . Quantification using peak areas of UV absorbance gave a nectar concentration at an average of 14.66 mmol l−1 (3518 ppm) unedone . Crop contents of B. terrestris gynes foraging on A. unedo had an average of 9.15 mmol l−1 (2195 ppm) unedone .Unedone, previously characterized from us honey , was fouTilia tomentosa nectar contained tiliaside. Tiliaside was verified by comparison with a pure standard isolated from Tilia honey  and [M + H]+ ), as well as the matching MS2 spectrum . Quantification using UV absorbance (308 nm) peak area gave an average concentration of 16.74 mmol l−1 . This is similar to the 11.86 mmol l−1 (6000 ppm = 0.6%) of tiliaside reported by Frérot et al. [Tilia cordata or Tilia platyphyllos).see also ) and acct et al.  in Swiss (b)Bombus terrestris gynes feeding on 3.79 mmol l−1 (910 ppm) unedone from A. unedo had lower infection levels in faeces samples 7 days after inoculation compared with the sugar water-fed control group  and were less likely to have developed an infection . Tiliaside from Tilia nectar reduced C. bombi infection levels in faeces of B. terrestris workers with pre-established C. bombi infections after feeding on 9.88 mmol l−1 (5000 ppm) tiliaside for 7 days, compared with the sugar water-fed control group .B. terrestris workers. The proportion of the corresponding aglycone, 4-(1-hydroxy-1-methylethyl)-1,3-cyclohexadiene-1-carboxylic acid , to tiliaside was low in the crop  and midgut , but significantly increased in the hindgut  and faeces  .HR-ESI-MS analyses showed that tiliaside was partially deglycosylated during gut passage in +m/z = 403.1960, Δppm −0.617 versus expected for C19H31O9; for MS2 spectrum see electronic supplementary material, figure S9), consistent with a hexoside of unedone, was recorded in an extract of faeces from bumblebee gynes fed on diets containing unedone. NMR spectroscopy of the compound purified from faeces of B. terrrestris gynes fed on unedone showed it to be a previously undescribed compound, unedone-8-O-β-d-glucoside . The structure of the compound was determined as a mono-glycosylated derivative of the known unedone [1H, 13C and DEPT), and 2D  NMR spectroscopic analysis and MS. The 13C NMR data for unedone [δC 82.3 (unedone gives δC 72.1). The analysis of the HMBC  showed that the C-8 position was substituted with a glycosidic moiety, and comparison of the reported data for β-glucose [1H-1H ROESY allowed the assignment of the relative configuration of the compound, with an epoxy group on one side and the hydroxy group and glucose groups on the other face of the molecule, as shown in electronic supplementary material, figure S3, and the compound was determined to be unedone-8-O-β-d-glucoside.A peak with similar UV absorbance to unedone (peak 245 nm) but higher mass C. bombi at 3.125 and 6.25 mmol l−1 (considerably below the average of 14.66 mmol l−1 measured in nectar) (a). We estimated the ED50 for unedone at 3.94 mmol l−1. By contrast, unedone-8-O-β-d-glucoside showed no inhibition of C. bombi in vitro up to a concentration of 6.25 mmol l−1, the highest concentration that was feasible for us to measure. This suggests glycosylation of unedone in the bumblebee gut will remove its antiparasitic activity. Tiliaside from linden nectar had no activity against C. bombi up to 20 mmol l−1 (higher than the concentration found in Tilia nectar by us), while the corresponding aglycone of tiliaside reduced C. bombi growth at concentrations from 5 to 20 mmol l−1 with an estimated ED50 of 6.35 mmol l−1 (b). Conversion of the approximately 17 mmol l−1 tiliaside in nectar into the corresponding aglycone would therefore lead to strong inhibition of C. bombi.Unedone inhibited  nectar) a. We estmmol l−1 b. Conver (d)6 CFU, and males 5.9 × 106 CFU per hindgut on BHI agar, with all individuals showing microbial growth. Hindguts of microbiome-depleted males and workers had no microbial growth on BHI agar, even when plated out at the lowest dilution step , except for a single individual each of the males and workers having 1 CFU at the lowest dilution step (possible contaminant). This suggests that our experimental protocol was effective for reducing microbial colonization of the gut and restoring it in the microbiome-inoculated individuals.Microbiome-inoculated bumblebee workers had an average of 1.9 × 10a). By contrast, in both workers and males, the unedone-8-O-β-d-glucoside was highly dominant (greater than 90%) over unedone in the midgut, suggesting that unedone is glycosylated in this gut compartment (a). No difference was apparent between microbiome-depleted and microbiome-inoculated individuals, suggesting that the glycosylation likely derives from enzymes of the bumblebee secreted into the midgut. A reversion to a higher proportion of unedone to unedone-8-O-β-d-glucoside was recorded in the hindgut of microbiome-inoculated males and workers, suggesting partial deglycosylation in this gut compartment. However, the reversion to unedone was not apparent in microbiome-depleted males and limited in microbiome-depleted workers (a), suggesting that the deglycosylation of unedone-8-O-β-d-glucoside was caused by the hindgut microbiome. A schematic of the fate of unedone in the gut is given in figure 3b.Only unedone, and not its glucoside, was detected in the crop of both workers and males, independent of microbiome status a. By conpartment a. No dif workers a, suggesc). Neither workers nor males showed apparent differences in the proportion of tiliaside to its aglycone between microbiome-inoculated and microbiome-depleted individuals (c), implying that the microbiome did not play a major role in this conversion, but that it is likely host-induced.The proportion of tiliaside to its aglycone increased from the crop to the midgut and hindgut c. Neitheividuals c, implyi. 4C. bombi. Importantly, the activity of secondary compounds can be both increased and decreased during gut passage, showing that a better understanding of the fate of nectar or pollen secondary metabolites after ingestion is necessary to determine their effects on parasites of pollinators. Simplified studies of secondary metabolites from pollen or nectar in in vitro assays alone may therefore not accurately predict their interactions and effects on parasites in the host, as they may either over- or underestimate effects. Our findings furthermore show that the antiparasitic activity of dietary secondary metabolites in a host can be altered by chemical changes induced by either the host or the resident microbiome. The effect of antiparasitic secondary metabolites on host infections will therefore likely depend on factors like the host genetic background (e.g. regarding enzymes processing secondary compounds), differences in host gene expression of enzymes metabolizing secondary compounds, gut pH [We demonstrate that the conversion of nectar secondary metabolites in the gut of bumblebees can modulate their activity against the common gut parasite , gut pH  and the C. bombi were driven by changes in glycosylation: for both Arbutus and Tilia nectar metabolites, aglycones had higher activity than corresponding glycosylated compounds. Similarly, Tasdemir et al. [in vitro activity of flavonoid aglycones against several human pathogenic trypanosomatid species than of corresponding glycosides. Glycosylation increases polarity and therefore water solubility of secondary metabolites but decreases their ability to cross cell membranes [C. bombi cell membranes relative to their corresponding glycosides. Once in the cytosol of the parasite, the secondary compounds will be able to interfere with cellular processes of the parasite, although exact mechanisms and cellular targets were not studied here. Our findings suggest that antiparasitic effects of glycosylated nectar or pollen metabolites will be underestimated if they can undergo deglycosylation in the host. Tiliaside from linden tree nectar was deglycosylated to the same extent in microbiome-depleted and microbiome-inoculated bumblebees, suggesting that this transformation was dependent on the host, not the microbiome. Bees produce β-glucosidases to catalyse the cleavage of β-glycosidic bonds in the crop and midgut [O-β-d-glucoside, deglycosidation in the hindgut was increased in microbiome-inoculated bumblebees, suggesting a role of the microbiome in this case.Differences in activity against r et al.  found geembranes . Therefod midgut , which aet al. [Apis mellifera) colonized by single strains from the major bacterial phylotypes of the resident microbiome of corbiculate bees suggest a dominant role of lactobacilli , including glycosidase hydrolases that could cleave sugars from glycosides [O-β-d-glucoside observed in our experiment in the hindgut of bumblebees, but additional experiments are needed to verify this. Both Lactobacillus and Bifidiobacterium genomes show considerable variation in glycosidase hydrolase contents between strains [More work is needed to understand which members of the microbiome in bees play key roles in the metabolic conversion of secondary metabolites, but experiments by Kešnerová et al.  on honeyn et al.  and bifiycosides . Bifidobycosides . Lactoba strains ,44. This strains ,46, coulet al. [p-coumaric acid upregulated the expression of UGTs in honeybees, consistent with their role in secondary metabolite detoxification in this bee species. Health trade-offs for pollinators between the benefits of detoxifying harmful secondary metabolites in the gut by glycosylation and the reduction of antiparasitic activity of glycosylated compounds as suggested in this study are plausible and deserve further attention. Under increased parasite pressure in the host environment, reduced glycosylation rates may be beneficial to limit parasite infections, as benefits from reduced parasite loads may outweigh the costs of damage from toxic secondary metabolites. Whether pollinators can fine-tune glycosylation in this manner in response to parasite risks remains to be investigated.Conversely, pollinators able to glycosylate secondary metabolites in the gut may have reduced antiparasitic benefits from them . To et al.  found thA. unedo) are the major autumn food source around the Mediterranean for B. terrestris [C. bombi infections have high fitness costs for B. terrestris gynes [C. bombi by unedone from A. unedo may improve their chances for winter survival and successful nest establishment in the following season. Linden trees (Tilia spp.) are major nectar sources in urban environments and temperate deciduous forests [Lastly, both tree species studied here have major ecological significance as food plants for bumblebees. Strawberry trees (rrestris , but alsrrestris . C. bombis gynes , and the forests –54. Our  forests  and thro forests ,55. Howe forests ,56.. 5in vitro studies, or studies of a single host–parasite system. Rather, an integrative view of the interaction of the hosts, parasites, secondary metabolites and the resident gut microbiomes needs to be taken for a fuller understanding of the potential benefits of floral reward phytochemicals on pollinator health.We show that the antiparasitic activity of nectar secondary metabolites can be both increased and decreased during gut passage in a common bumblebee pollinator. This modulation of antiparasitic activity can derive both from the host and from the resident gut microbiome. Effects of secondary metabolites on pollinator parasites therefore cannot necessarily be extrapolated from


The median age is 6th decade with a significant female preponderance.We describe gross and histopathological findings of mucinous tubular and spindle cell carcinoma in a 65-year-old male patient. He presented with right abdominal pain for one month. The pain was dull and mild to moderate in intensity. On contrast-enhanced ultrasound, a well-defined, smoothly marginated heterogeneous hyperechoic lesion measuring 5.5x6cm was noted at the upper pole of the right kidney. The lesion was hyper enhancing compared to the rest of the renal parenchyma. Based on the radiological findings, possibilities of chromophobe RCC and oncocytoma were considered. The abdominal computed tomography (CT) revealed a hyperdense lesion in the upper pole of the right kidney with a relatively well-defined margin measuring 63x65x69mm with no evidence of significant post-contrast enhancement in the either arterial, venous or delayed phase. The patient underwent a right radical nephrectomy. On gross examination, a well-encapsulated mass was seen wholly occupying the upper pole of the kidney measuring 6.9x6.5x5.5cm. The cut surface was variegated with grey-white, firm areas admixed with mucinous and hemorrhagic foci . No capsMicroscopically, the tumor was well-demarcated from the adjacent native renal parenchyma . It was


Minimal change disease (MCD) is the most common pathological subtype of pediatric idiopathic nephrotic syndrome (INS). It has been suggested that IgM deposition might predict kidney function deterioration in the course of MCD. However, the specific role of IgM deposition in the prognosis of MCD is still controversial. This study aims to investigate the clinical significance of IgM deposition on delayed remission and early relapse in a pediatric population.This study enrolled 283 children diagnosed with MCD by renal biopsy in a single center from 2010 to 2022. These cases were divided into two groups according to the histopathological deposition of IgM. Patients' demographics, clinical parameters, and follow-up data were collected and analyzed. The primary and secondary outcomes were defined as the time to the first remission and the first relapse.P = 0.001), and showed more recurrent cases  compared with the IgM-negative group. The Kaplan-Meier analysis showed that the IgM-positive group had a lower cumulative rate of the first remission  and a higher rate of the first relapse  than the IgM-negative group. Multivariate Cox analysis showed that IgM deposition was independently associated with the delayed first remission  and the early first relapse .The IgM-positive group had a weaker response to steroids (steroid-sensitive: 23.5% vs. 40.8%; steroid-dependent: 74.0% vs. 51.0%; steroid-resistant: 18.4% vs. 8.2%, IgM deposition was associated with a weaker steroid response. MCD children with IgM deposition were prone to delayed first remission and early first relapse. Primary glomerular disease in children commonly presents as nephrotic syndrome (NS), characterized by massive proteinuria, hypoproteinemia, and peripheral edema. NS includes a variety of pathological subtypes, of which MCD accounts for about 70%–90% of children >1 year of age . HoweverThe role of IgM deposition in the progression and prognosis of MCD is controversial. A report in 2019 showed that IgM deposition was associated with hypertension, steroid dependence, and Chronic kidney disease (CKD) in children with MCD, which may be a predictor of deterioration of renal function . Sandra In this retrospective study, children (≤18 years) with MCD diagnosed by kidney biopsy from 2010 to 2022 were recruited from Jinling hospital. We enrolled cases who met the following criteria: (1) 24-h urine protein to creatinine ratio (UP/CR) ≥0.2 g/g; (2) patients with MCD whose biopsies were performed at any age between 1 and 18 years. Individuals with one of the following conditions were excluded: (1) infectious diseases ; (2) individuals of contemporary autoimmune disease, diabetes mellitus, malignant tumors; (3) drug-induced NS; (4) missing data on IgM deposition in renal pathology; (5) the glomerular number on light microscopy <10.All clinical and pathological data were obtained retrospectively from medical records. The baseline investigations were taken during the kidney biopsy. Follow-up data, including treatment, steroid response, time to the first remission and relapse, was collected and analyzed at 1, 2, 3, 6, 12 months and longer follow-up. The last follow-up time was estimated from the biopsy to the previous encounter in our center. The diagnosis of hypertension was based on the 2017 AAP Blood Pressure Clinical Practice Guidelines .2/day or 2 mg/kg/day) and no relapse during corticosteroid therapy. (4) Steroid-resistant was defined as a lack of CR for at least 4 weeks of full-dose oral prednisone. (5) Steroid-dependent was defined as follows: (1) Reach CR after the 4 weeks of full-dose oral corticosteroid; (2) Two consecutive relapses during corticosteroid therapy or within 15 days of discontinuing corticosteroid therapy; (3) Infectious factors should be excluded at the same time; (4) When relapse occurs, patients could reach CR once the corticosteroid dose is increased (either at full dose or beyond the relapse dose).(1) IgM-positive was defined as the intensity of the IgM staining by immunofluorescence ≥1+. (2) Grade of tubulointerstitial injury scored as none (<10%), mild (10%–24%), moderate (25%–49%), and severe (≥50%), with the percentages mentioned in parentheses. The change of chronic tubulointerstitial injury (CTI) indicated tubular atrophy/interstitial fibrosis. The manifestation of acute tubulointerstitial injury (ATI) was interstitial edema, inflammatory cellular infiltration, and necrosis in tubular epithelia. (3) Steroid-sensitive was defined as CR after the 4 weeks of full-dose oral corticosteroid  was a decrease in 24-h UP/Cr to <2 g/g but >0.2 g/g (or >20 and <200 mg/mmol) with a 50% reduction from its peak value, or the test result of the dipstick decreases by at least 1+, but it does not reach negative. CR was a decrease in 24-h UP/Cr ≤0.2 g/g (or 20 mg/mmol or negative or trace dipstick) on three or more consecutive occasions. Remission was the achievement of PR or CR. Relapse was defined as an increase in UP/Cr to ≥2.0 g/g in cases with a 50% increase from its valley value or in urine dipstick to ≥3+after the remission. Frequent relapse was defined as ≥2 relapses per 6 months or ≥4 relapses per 12 months.This cohort study's primary and secondary endpoints were the first remission and the first relapse, respectively. When patients achieved the first remission after biopsy, we considered this the first remission. And the time interval from treatment to the first day of remission would be recorded. Time to the first relapse was the time interval from the first remission to the occurrence of the first relapse.U test, represented by the median , while categorical variables using the chi-square test or Fisher exact test were expressed as frequency (percentage). All data were tested two-sided, with P < 0.05 regarded as statistically significant.The SPSS 26.0, Graph Pad Prism 8.0, and Adobe Illustrator 2020 were used for statistical analysis in this cohort study. The continuous variables were compared between groups using the Mann-Whitney P > 0.05). No severe ATI and moderate to severe CTI were observed in the renal pathology of all MCD individuals.As shown in P < 0.001). In immunofluorescence, 16 (11.8%) patients also had positive C3 deposition in the IgM-positive group, while only 7 (4.8%) were in the IgM-negative group (P = 0.048). The IgM-positive group had a lower level of serum albumin  and a higher level of eGFR  than the IgM-negative group.The median age at the renal biopsies of patients was 12.6 years old for the IgM-positive group and 15.2 years for the IgM-negative group (P = 0.025) than the IgM-negative group. More details of immunosuppressive therapy for the steroid-sensitive nephrotic syndrome (SSNS), steroid-dependent nephrotic syndrome (SDNS) and steroid-resistant nephrotic syndrome (SRNS) patients are shown in A summary of treatment and outcomes is shown in P = 0.001). For instance, the IgM-positive group has fewer steroid-sensitive patients (23.5% vs. 40.8%), more steroid-dependent (74% vs. 51%) and steroid-resistant patients (18.4% vs. 8.2%) than the IgM-negative group. 283 (100%) patients reached the first remission during a median follow-up time of 30 days. The median time to the first remission was significantly different between the two groups . 171 (60.8%) patients relapsed at least once during a median follow-up time of 200 days. Although the frequencies of frequent relapses (20.6% vs. 19.7%) and infrequent relapses (44.9% vs. 36.1%) were more common in the IgM-positive group than in the IgM-negative group, there was no statistically significant difference in relapse between the two groups (P = 0.182).The IgM-positive group was less responsive to steroid therapy than the IgM-negative group  (P < 0.001), serum albumin , steroid-resistant  were associated with the first remission. The result of the multivariate Cox regression analysis is listed in P = 0.001), serum albumin , steroid-dependent  and steroid-resistant  were independently association with the first remission.The Kaplan-Meier survival analysis showed that the IgM-positive group had a lower cumulative rate of the first remission than the IgM-negative group  . The 1-,P > 0.05) (P = 0.090) and multivariate analysis  showed that IgM deposition was not associated with the first relapse. Serum albumin  and steroid-dependent  were independently associated with the first relapse in the univariate Cox analysis. In the multivariate Cox analysis, serum albumin  and steroid-dependent  were still independently associated with the first relapse.However, there is no statistical difference in relapses between the positive and negative groups  . MoreoveP = 0.047). P = 0.034). The 3-month, 6-month, 1-year and 2-year remission rates were 12.98% and 18.87%, 21.37% and 30.19%, 34.35% and 41.17%, 34.35% and 47.17% for the IgM-negative group and the IgM-positive group, respectively. The univariate Cox regression analysis showed IgM deposition  and serum albumin  were associated with the first relapse. P = 0.045) and serum albumin  are independently associated with the first relapse.We suggested that a significant portion of patients relapsed after a follow-up of 1 year. Therefore, extended follow-up may be necessary to demonstrate the significance of IgM deposition in relapse. Finally, 237 patients with a follow-up of more than 1 year were included to detect the predictors of the first relapse. The results are shown in Several studies have examined the clinical characteristics and renal outcomes in pediatric MCD with or without IgM deposition , 9, 10. In previous studies, MCD children with IgM deposition showed a more common frequency of steroid dependence , 11. A sIn our study, the time to the first remission was significantly longer in the IgM-positive group than in the IgM-negative group. And the IgM deposition was an independent risk factor for delayed remission. Nevertheless, Mateja et al. found no differences in the time to the first remission and frequency of the first remission between MCD children with and without IgM deposition . An adulA study published in 2021 indicated that IgM mesangial deposition was a risk factor for relapses of adult-onset MCD . In our IgM deposition can be observed in some MCD and focal segmental glomerulosclerosis (FSGS) patients' renal biopsy samples under immunofluorescence , 17. MCDde novo antigens exposed to the glomerulus, activate the complement system, and further contribute to renal injury (IgM is the largest serum antibody in the human body and the initial activator for the complement cascade . Some scl injury , 23. WheThere were some limitations to our study. First, our study still had the inevitable drawbacks of a retrospective cohort design, indicating that the patients needed to accept uniform treatment. And we did not adjust the immunosuppressive therapy in the Cox regression model. However, it should be noted that there was no difference between patients with and without IgM deposition using ACEI/ARB or steroid or immunosuppressive agents, as shown in MCD children with IgM deposition are more likely to present with steroid dependence or steroid resistance, suggesting a possible association with a poorer disease state. IgM deposition is an independent risk factor for delayed remission and an independent promoting factor for early relapse of pediatric MCD. Therefore, enhanced disease activity surveillance should be considered in MCD pediatric patients with IgM deposition. Nonetheless, a prospective study is needed to verify the function of IgM deposition in MCD outcomes.